In breeding of pigs a major problem is to keep the new-born, and in particular post weaning young pigs, disease-free. In that respect intestinal disorders are among the most widespread and serious problems, and swine breeding and production farms all over the world suffer sizeable losses of livestock each year from outbreak of these diseases. Further losses arise from the costs of medication, growth retardation and other consequences of the diseases, which may be more substantial than direct damage due to mortality.
It is known that the causative agents responsible for a significant number of the diarrhoeas in young pigs are enterotoxigenic strains of E. coli ETEC. Several ETEC have been isolated from infected animals and types of E. coli strains, such as E. coli F18, and E. coli F4 (formerly known as K88), are among the major lethal strains found in young pigs.
The designations F18 and F4 refer to fimbriae types of the ETEC and are used to distinguished the different strains. The adhesive fimbriae mediate the colonisation of E. coli in the intestine. Colonisation depends on the adherence of the bacteria to the enterocytes and subsequent proliferation and toxin production of the ETEC will cause the diarrhoea.
The disease develops after the ETECs are introduced into the intestinal tract of the piglets. The ETEC bacteria adhere to the wall of the small intestine through their surface protein antigens (fimbriae), multiply there in large numbers and transfer their toxins directly to the intestinal epithelial cells. Due to the effect exerted by the toxins, the fluid-absorbing activity of intestinal epithelial cells will cease and the cells will secrete a large volume of fluid into the intestinal lumen resulting in the development of more or less severe diarrhoeas.
In the art, efforts have been made in order to control diarrhoea in animals, such as pigs, caused by ETEC. U.S. Pat. No. 4,443,549 describes a method of producing monoclonal antibodies against adhesion of a pathogenic bacterium, which adhesion mediates attachment of the bacterium to mucocutaneous tissue. The antibodies may be used in a pharmaceutical composition suitable for oral administration to animals.
U.S. Pat. No. 4,761,372 discloses a plasmid comprising genes coding for an immunogenic, non-toxic, heat labile enterotoxin and/or a non-toxic, heat stable enterotoxin and an E. coli containing this plasmid for use as a live vaccine for vaccinating humans and animals against diarrheal diseases.
Additionally, WO 00/58476 describes a method of producing a live, orally applicable E. coli vaccine for the prevention of postweaning diarrhoea in pigs. An enterotoxin-free strain of E. coli which produces two adhesive fimbriae (F4 and F18) are administered to the young pigs in an attempt to provide local protection.
However, not all pigs succumb to E. coli infections. Susceptibility to adhesion, i.e. expression of receptors in pigs for binding of particular fimbriae, has been shown to be genetically controlled by the host. The mechanism for resistance appears to be that intestinal borders in resistant animals are not colonised by E. coli i.e., the bacteria do not adhere to the intestinal walls of resistant animals.
An attempt to detect E. coli resistance is reported in WO 98/53101. The application relates to a method for differentiating between pigs that are either resistant or susceptible to F18 E. coli related diseases. The differentiation is performed using a DNA test for DNA polymorphisms in the alpha (1,2) fucosyltransferase 1 (FUT1) gene on the porcine chromosome 6.
Enterotoxigenic Escherichia coli cells (ETEC) that expresses the F4ab or F4ac fimbriae (formerly know as K88ab and K88ac) are major causes of diarrhoea and death in neonatal and young pigs (Wilson and Francis, 1986). In Denmark, ETEC F4 is present in about 25% of the reported diarrhoea cases (Ojeniyi et al., 1994).
In 1975 Sellwood and co-workers published a paper describing the existence of two pig phenotypes in relation to ETEC F4, namely resistant pigs and susceptible pigs, respectively. In 1977, Gibbons et al. showed that ETEC F4ac resistance was inherited as an autosomal recessive Mendelian trait and linkage to the transferrin locus (TF) was suggested and later confirmed (Guérin et al. 1993). Linkage mapping of the porcine loci responsible for susceptibility towards ETEC F4ac and F4ab led to the hypothesis that a potential candidate gene was located on pig chromosome 13 (Edfors-Lilja et al., 1995), but no candidate gene was found or suggested. This means that the only available diagnostic test for this type of ETEC F4 resistance is the adhesion test developed by Sellwood et al. in 1975. Since the adhesion test either demands major intestinal surgery or slaughter of the pig and the fact that it is very laborious makes it difficult to incorporate it into breeding programs.
The prerequisites that need to be fulfilled in order to incorporate a diagnostic test into a breeding program is that it needs to be quick, easy to use and allowing precise genotyping of live animals; however, so far no suitable method fulfils this need.